| ObjectiveThis research through the plasmid transfection technology regulation BAG3 protein expression in colon cancer cell lines HCT116 and RKO,and then to explore the regulation of BAG3 protein expression after the influence of the drug sensitivity to 5-fu,aims to address BAG3 protein in its role in the development of colon cancer,looking for new drug targets for multiple drug resistance in colon cancer.Methods1.In vitro culture of colon cancer cell lines HCT116 and RKO,in which BAG3 protein was overexpressed by the transient transfection of exogenous pcDNA3-N-EGFP BAG3(WT)into the two colon cancer cell lines.The BAG3 was silenced by the transfection of shBAG3 into the two colon cancer cell lines.The BAG3 protein overexpression and silencing cell model were successfully constructed by Q-PCR and Western blot experimental methods from mRNA and protein levels.2.The effects of overexpression BAG3 or knockdown of BAG3 on the drug sensitivity of colon cancer cell lines to 5-fu(5-fluorouracil)were studied by using MTT assay to detect cell proliferation,coordinate fixed-point scratch repair experiment and transwell transfer compartment experiment to detect cell migration and invasion and flow cytometry to detect cell apoptosis and other experimental methods.Rerults1.After Q-PCR and Western blot detection,the colon cancer cells HCT-116 and RKO transfected exogenous pcDNA3-N-EGFP BAG3(WT),compared with the control group(transfected PCDNA3-N-EGFP empty plasmid),The expression of BAG3 in RNA level and protein level was enhanced,the difference was significant;the colon cancer cells HCT-116 and RKO transfected different knockdown loci shBAG3,we found that the BAG3 knockdown effect of shBAG3 transfected with shBAG3-1334 was the best compared with that of control group(shNC).The cell model was successfully constructed and used in subsequent experiments.2.Under different concentrations of 5-fu,overexpression of BAG3 increased cell viability,migration and invasion,and decreased the number of apoptotic cells,indicating that overexpression of BAG3 promoted the resistance of colon cancer cells to 5-fu.It inhibited the apoptosis of colon cancer cells and increased the resistance of colon cancer cells to 5-fu.3.Under different concentrations of 5-fu,the cell viability,migration ability,invasion ability were decreased and apoptotic cells increased after BAG3 knockdown,which indicated that knockdown BAG3 promoted the apoptosis of colon cancer cells and enhanced the drug sensitivity of colon cancer cells to 5-fu.ConclusionsOverexpression BAG3 promted the resistance of colon cancer cells to 5-fu,and knockdown of BAG3 enhanced the drug sensitivity of colon cancer cells to 5-fu and BAG3 may be a new biological target for chemotherapy resistance in colon cancer. |
